A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing.

Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.

3D bioprinting ofE. coliMG1655 biofilms on human lung epithelial cells for building complexin vitroinfection models.

Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complexin vitromodels that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robustin vitroinfection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal forEscherichia coliMG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complexin vitroinfection models comprising bacterial biofilms and human host cells.

The inherent antibiotic activity of myxobacteria-derived autofluorescent outer membrane vesicles is switched on and off by light stimulation.

Outer membrane vesicles are small, lipid-based vesicles shed from the outer membrane of Gram-negative bacteria. They are becoming increasingly recognised as important factors for resistance gene transfer, bacterial virulence factors and host cell modulation. The presence of pathogenic factors and antimicrobial compounds in bacterial vesicles has been proven in recent years, but it remains unclear, if and how environmental factors, such as light specifically regulate the vesicle composition. We report the first example of autofluorescent vesicles derived from non-pathogenic soil-living myxobacteria. These vesicles additionally showed inherent antibiotic activity, a property that is specifically regulated by light stimulation of the producing bacteria. Our data provide a central basis for better understanding the environmental impact on bacteria-derived vesicles, and design of future therapeutic options.

Transferring Microclusters of P. aeruginosa Biofilms to the Air-Liquid Interface of Bronchial Epithelial Cells for Repeated Deposition of Aerosolized Tobramycin.

As an alternative to technically demanding and ethically debatable animal models, the use of organotypic and disease-relevant human cell culture models may improve the throughput, speed, and success rate for the translation of novel anti-infectives into the clinic. Besides bacterial killing, host cell viability and barrier function appear as relevant but seldomly measured readouts. Moreover, bacterial virulence factors and signaling molecules are typically not addressed in current cell culture models. Here, we describe a reproducible protocol for cultivating barrier-forming human bronchial epithelial cell monolayers on Transwell inserts and infecting them with microclusters of pre-grown mature Pseudomonas aeruginosa PAO1 biofilms under the air-liquid interface conditions. Bacterial growth and quorum sensing molecules were determined upon tobramycin treatment. The host cell response was simultaneously assessed through cell viability, epithelial barrier function, and cytokine release. By repeated deposition of aerosolized tobramycin after 1, 24, and 48 h, bacterial growth was controlled (reduction from 10 to 4 log10 CFU/mL), which leads to epithelial cell survival for up to 72 h. E-cadherin's cell-cell adhesion protein expression was preserved with the consecutive treatment, and quorum sensing molecules were reduced. However, the bacteria could not be eradicated and epithelial barrier function was impaired, similar to the currently observed situation in the clinic in lack of more efficient anti-infective therapies. Such a human-based in vitro approach has the potential for the preclinical development of novel anti-infectives and nanoscale delivery systems for oral inhalation.

Genome-Guided Discovery of the First Myxobacterial Biarylitide Myxarylin Reveals Distinct C-N Biaryl Crosslinking in RiPP Biosynthesis.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C-N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C-C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.

Satisfacción con el servicio de posortodoncia de la Universidad Santo Tomás según métricas Lean-Six-Sigma / SatisfactionService Post-Orthodontic University Santo Tomas According ToLean-Six-Sigma Metrics

Introducción: La metodología Lean-Six-Sigma es utilizada actualmente en el área de la salud con el objeto de mejorar la calidad y competitividad de los servicios; pero aún no se tienen reportes de su uso en el área de ortodoncia. Objetivo: Determinar la satisfacción posortodoncia por medio de niveles sigma de los pacientes que asisten a una clínica docente-asistencial en Bucaramanga, Colombia. Métodos: Estudio observacional, descriptivo, transversal. La población estuvo constituida por 100 pacientes de ambos sexos, que ya habían concluido el tratamiento de ortodoncia. Se seleccionó toda la población que hubiera finalizado el tratamiento entre julio de 2017 y junio 2018 de acuerdo con criterios de inclusión y exclusión. Se realizó una encuesta telefónica de 17 preguntas y siete dimensiones de satisfacción. La confiabilidad del cuestionario se evaluó con la consistencia interna del cuestionario mediante el alpha de Cronbach en el que se obtuvo 0,87, y para la validez se utilizó el método de Lawshe. Se usó la metodología Lean-Six-Sigma para evaluar la calidad de la satisfacción; se presentan los resultados en porcentaje y niveles sigma. Resultados: La calidad en satisfacción en el servicio fue de 91,27 por ciento (medido convencionalmente) y 1,36 sigmas; en el grupo de 16-19 años fue de 93,6 por ciento y 1,52 sigmas; y para el grupo de más de 27 años, 84,9 por ciento y 1,03 sigmas. El sexo femenino reportó un 88,57 por ciento y 1,2 sigmas, mientras que el masculino, 93,46 por ciento y 1,51 sigmas. Conclusiones: Los resultados de satisfacción en ortodoncia, medidos por métodos convencionales, mostraron porcentajes aceptables, pero mostraron resultados incompetentes al medirse con niveles sigma, lo cual indica que existen problemas ocultos por identificar y que corresponderían a una siguiente investigación(AU)

Introduction: The Lean-Six-Sigma methodology is currently used in the health area in order to improve the quality and competitiveness of services; but there are still no reports of its use in the orthodontic area. Objective : to determine the post-orthodontic satisfaction by means of sigma levels of the patients who attend a teaching-assistance clinic in Bucaramanga, Colombia. Methods: Observational descriptive cross-sectional study. Population: 100 patients (female and male) who finish orthodontic treatment. Sample selection method: the entire population that had completed the treatment between July 2017 and June 2018 was selected according to inclusion and exclusion criteria. A telephone survey of 17 questions and seven dimensions of satisfaction was carried out. The reliability of the questionnaire was evaluated with the internal consistency of the questionnaire by means of the Cronbach's alpha in which 0.87 was obtained, and the Lawshe method was used for validity. The Lean-Six-Sigma methodology was used to assess the quality of satisfaction; Results are presented in percentage and sigma levels. Results: The quality of service satisfaction was 91.27 percent (conventionally measured) and 1.36 sigmas; in the group of 16 to 19 years it was 93.6 percent and 1.52 sigmas; and for the group over 27 years old, 84.9 percent and 1.03 sigma. The female sex reported 88.57 percent and 1.2 sigmas, and the male reported 93.46 percent with 1.51 sigmas. Conclusions: The satisfaction results in orthodontics, measured by conventional methods, showed acceptable percentages, but showed incompetent results when measured with sigma levels, which indicates that there are hidden problems to be identified and that they would correspond to a subsequent investigation(AU)

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners.

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.

Mercaptothiacalixarenes Steer 24 Copper(I) Centers to form a Hollow-Sphere Structure Featuring Cu2 S2 Motifs with Exceptionally Short Cu⋠⋠⋠Cu Distances.

Tetramercaptotetrathiacalix[4]arene (LH4 ) can be used as a coordination platform to bind four CuI ions at the thiolate and thioether S atoms. Donor ligands such as phosphanes can stabilize the resulting [LCu4 ] units, which then remain monomeric ([(Ph3 PCu)4 L]). In the absence of donor ligands, they aggregate, providing a hexamer ([LCu4 ]6 ) in high yields, with a hollow-sphere structure formed by an unprecedented Cu24 S48 cage that is surrounded by the organic framework of the calixarene chalices. Preliminary NMR experiments with regard to the host-guest chemistry in solution showed that the compound represents a polytopic host for acetonitrile and methane.

Bismuthanes as Hemilabile Donors in an O2 -Activating Palladium(0) Complex.

A xanthene-based bismuthane/phosphane chelating ligand has been accessed that has enabled the synthesis of a palladium(0) bismuthane complex. The bismuthane donor proved to be hemilabile as it switched to a dangling position upon addition of O2 that gave a palladium(II) peroxide complex. Unlike the corresponding 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) palladium peroxide, the bismuth analogue could be employed for catalytic phosphane oxidation and oxidative phenol coupling.

Activation of Dioxygen at a Lewis Acidic Nickel(II) Complex: Characterization of a Metastable Organoperoxide Complex.

In metal-mediated O2 activation, nickel(II) compounds hardly play a role, but recently it has been shown that enzymes can use nickel(II) for O2 activation. Now a low-coordinate Lewis acidic nickel(II) complex has been synthesized that reacts with O2 to give a nickel(II) organoperoxide, as proposed for the enzymatic system. Its formation was studied further by UV/Vis absorption spectroscopy, leading to the observation of a short-lived intermediate that proved to be reactive in both oxygen atom transfer and hydrogen abstraction reactions, while the peroxide efficiently transfers O atoms. Both for the enzyme and for the functional model, the key to O2 activation is proposed to represent a concomitant electron shift from the substrate/co-ligand.

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.

Phosphine-Stabilized Borylenes and Boryl Anions as Ligands? Redox Reactivity in Boron-Based Pincer Complexes.

Stabilized borylenes (L2 BH:) with weakly π-accepting substituents L, such as phosphines, were previously believed to be unstable. In the current manuscript, we describe a series of complexes formally containing a phosphine-stabilized borylene or boryl anion. In contrast to common trivalent boron compounds, the boron-based ligands in this study act as electron-donating ligands. The reported iron hydride complexes exhibit a unique reactivity pattern, undergoing a reversible B-H reductive elimination concomitant with oxidation of the boron(I) center.

Trapping Aluminum Hydroxide Clusters with Trisilanols during Speciation in Aluminum(III)-Water Systems: Reproducible, Large Scale Access to Molecular Aluminate Models.

To gain molecular level insights into the properties of certain functions and units of extended oxides/hydroxides, suitable molecular model compounds are needed. As an attractive route to access such compounds the trapping of early intermediates during the hydrolysis of suitable precursor compounds with the aid of stabilizing ligands is conceivable, which was tested for the aluminum(III)/water system. Indeed, trisilanols proved suitable trapping reagents: their presence during the hydrolysis of Al(i) Bu2 H in dependence on the amount of water used allowed for the isolation of tri- and octanuclear aluminum hydroxide cluster complexes [Al3 (µ2 -OH)3 (THF)3 (PhSi(OSiPh2 O)3 )2 ] (1) and [Al8 (µ3 -OH)2 (µ2 -OH)10 (THF)3 (p-anisylSi(OSiPh2 O)3 )4 ] (2). 1 can be regarded as the Al(OH)3 cyclic trimer, where six protons have been replaced by silyl residues. While 2 features a unique [Al8 (µ3 -OH)2 (µ2 -OH)10 ](12+) core. In contrast to most other known aggregates of this type, 1 and 2 can be readily prepared at reasonable scales, dissolve in common solvents, and retain an intact framework even in the presence of excessive amounts of water. This finding paves the way to future research addressing the reactivity of the individual functional groups.

The Effect of Substituents at Lewis Acidic Bismuth(III) Centers on Its Propensity to Bind a Noble Metal Donor.

To investigate the effect of X in ambiphilic compounds BiX(o-PPh2-C6H4)2, PBiP-X, on metallophilic Pt-Bi interactions in its PtCl2 complexes two new derivatives PBiP-Me and PBiP-C6F5 were synthesized. Reaction with dichloro(1,5-cyclooctadiene)platinum(II) led to the platinum(II) complexes [PtCl2(PBiP-Me)], 3, and [PtCl2(PBiP-C6F5)], 4, which together with the halide [PtCl2(PBiP-Cl)], 2, reported previously, establish a series of related PBiP-X complexes differing only in X. This could be complemented by accessing [PtCl2(PBiP-OTf)], 5, through the reaction of 2 with AgOTf. Analysis of the geometrical and electronic structures of these complexes revealed that in all cases the platinum(II) centers act as donors (through their filled d(z(2)) orbitals) to the bismuth(III) centers (possessing σ*(Bi-X)/6p acceptor orbitals). The strength of these interactions increases with increasing electron-withdrawing character of X, which supports the conceptual approach in constructing this new class of compounds.

Metal-ligand cooperation in H2 activation with iron complexes bearing hemilabile bis(diphenylphosphino)amine ligands.

The octahedral transition-metal complex [(dppa)Fe(Ph2P-N-PPh2)2] (1) [dppa = bis(diphenylphosphino)amine] with homofunctional bidentate ligands is described. The ligand exhibits hemilability due to its small bite angle and the steric repulsion of the coordinated donor groups. As the {Ph2P-N-PPh2}(-) ligand can act as an internal base, heterolytic cleavage of dihydrogen by complex 1 leads to the formation of the hydride complex [(dppa)(Ph2P-N-PPh2)Fe(H)(κ(1)-Ph2P-NH-PPh2)2] (2), representing an example of cooperative bond activation with a homofunctional hemilabile ligand. This study demonstrates that hemilability of homofunctionalized ligands can be affected by careful adjustment of geometric parameters.

The new NH-acid HN(C6F5)(C(CF3)3) and its crystalline and volatile alkaline and earth alkaline metal salts.

Herein we report on the new NH-acid N-(2,3,4,5,6-pentafluorophenyl)-N-nonafluoro-tert-butylamine, HN(C6F5)(C(CF3)3), bearing two different sterically demanding and strongly electron-withdrawing perfluorinated amine substituents. The title compound and seven of its alkaline and alkaline earth metal salts were synthesized and investigated concerning their thermal, spectroscopic, and structural properties. The Li, Na, K, Cs, and Mg salts were investigated by single-crystal XRD analysis. The molecular structures reveal interesting motifs such as manifold fluorine metal secondary interactions. The lithium and magnesium compounds exhibit a remarkable thermal stability and an unexpectedly high volatility. We believe that this report will provoke investigations to apply the corresponding anion in ionic liquids, in lithium electrolytes, and as a weakly electron-donating ligand in the preparation of highly Lewis-acidic main group, rare earth, or transition metal complexes.

Formation of an iron phosphine-borane complex by formal insertion of BH3 into the Fe-P bond.

A unique hydrido phosphine-borane iron(II) complex [(dppa)(Ph2P-N-P(BH3)Ph2)Fe(H)] (1) was obtained by the reaction of iron(II) chloride and two equivalents of bis(diphenylphosphino)amine (dppa) with an excess of sodium borohydride in acetonitrile-ethanol mixtures. Detailed investigations of the reaction revealed that a mixture of cis- and trans-[(dppa)2Fe(NCMe)2]²âº is formed prior to the reduction by sodium borohydride. Depending on the solvent, different products were obtained by the reduction: in acetonitrile-ethanol mixtures the hydrido phosphine-borane complex 1 is formed by formal insertion of BH3, while the reduction in pure acetonitrile results in the formation of the cationic complex trans-[(dppa)2Fe(H)(NCMe)](BH4) (4). Complex 4 is remarkably stable in ethanol and does not undergo phosphine-borane formation, even in the presence of excess sodium borohydride. This observation suggests that the phosphine-borane complex is generated by the reaction with the first equivalent of sodium borohydride with the participation of ethanol, followed by deprotonation or dihydrogen elimination. Experiments with similar diphosphine ligands, such as bis(diphenylphosphino)methane, did not yield a phosphine-borane complex, indicating the crucial role of the amine group in the observed reactivity.

A defect supertetrahedron naphthoxime-based [Mn(III)9] Single-Molecule Magnet.

A new [Mn(III)9] complex was synthesized from a naphthoxime-based ligand. It was structurally and magnetically characterized, revealing a rare defect supertetrahedral topology and promising SMM properties with a large energy barrier of 67 K.

Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease.

Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.

Laminin induced local axonal translation of ß-actin mRNA is impaired in SMN-deficient motoneurons.

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of ß-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the ß-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of ß-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that ß-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the ß-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of ß-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn(-/-);SMN2). Taken together our findings suggest that local translation of ß-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.